Inflammation Models in Skin Explants

In the dithranol inflammation model, a solution of 0.3% of 1,8,9-Anthracenetriol (dithranol) (Sigma-Aldrich, Merk Life Science S.r.l Milano, Italy) in acetone (w/v) was applied on the skin surface immediately after mounting the sample in the bioreactor chamber [22 (link)]. The time points chosen for analyzing the inflammatory effects were 1 h, 3 h, 6 h and 24 h. The treatment was not prolonged over 24 h because this is the classical dithranol therapy; after that, healthy skin surrounding the treated area shows irritation signs [21 (link)]; accordingly, preliminary tests on our skin explants showed that, at the concentration chosen for our study, dithranol induced severe tissue damage after 24 h.
In the substance P-inflammation model, the neuropeptide (Bachem AG, Bubendorf, Switzerland) was added to the medium at a concentration of 10 μM [25 (link)]. Analyses were performed not only at 24 h but also at 48 h and 72 h to monitor the inflammatory process at the classical pharmacokinetic time points used to test therapeutic treatments. Moreover, it is known that the effects of substance P administration in vitro may last for a long time, e.g., [67 (link),68 (link)].
The control (untreated) samples were maintained in the bioreactor under the same experimental conditions but avoiding dithranol or substance P treatment and were analyzed at the same time points.